Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Szexuális úton terjedő betegségekhez kapcsolódó preventív és gondozási feladatok – A szexuális úton terjedő betegségekről való ismeretek különböző életkorokban

Szakdolgozatomban részletesen foglalkozok a különböző életkorú csoportok szexuális úton terjedő betegségekhez kapcsolódó ismereteivel.BSc/BAápolás és betegellátás – szülésznőmagyarnappaliV

Szuggesztív kommunikáció a szülészeti ellátásban

A szakdolgozat témája a szuggesztív kommunikáció, melyről manapság kevés szó esik az egészségügyi ellátásban, specifikusan csak a pszichológia-pszichiátria területén fordul elő. Mivel szakterületem a szülészet-nőgyógyászati ellátás, szülésznőket és orvosokat vontam be vizsgálatomba abból a célból, hogy feltárjam: munkájuk során használják-e a meggyőzést vagy befolyásolást a beteg állapotának javulása vagy a fájdalom megélésének megváltoztatása céljából.MSc/MAegészségügyi tanármagyarlevelezőV

Hereditaer angioneuroticus oedema = hereditary angioedema

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Súlyosan égett beteg utógondozásának jelentősége

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Paradicsom tumorok mellett jelentkező hólyagos bőrtünetek

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Calcineurin, tubulin and 14-3-3 are the major proteins binding to TRESK-loop-His₈ in affinity chromatography experiments.

<p><b>A.</b> Mouse brain proteins, remaining on the columns after the NaCl gradient, were eluted with 7 M urea. Three fractions from the Ni-NTA control column (N1–N3) and from the column containing TRESK-loop-His₈ (T1–T3) were analyzed by SDS-PAGE and Coomassie Blue staining. The two intense bands from fraction T2 were identified by mass spectrometry as calcineurin and tubulin (as indicated in the table below the gel). <b>B.</b> TRESK-loop-His₈ (immobilized on Ni-NTA resin) was phosphorylated with protein kinase A (PKA) before the affinity chromatography. The phosphorylated bait protein interacted with different 14-3-3 isoforms (see band 3 and 4; P1 and P2 lanes represent two independent experiments).</p

Tubulin binds to GST fusion constructs containing long fragments of the cytoplasmic loop of human TRESK.

<p><b>A.</b> Schematic transmembrane topology of TRESK subunit is shown. The fragments of the intracellular loop (174–280, 204–280, 232–280, 174–231 and 174–247), which were fused to GST, are indicated with bars of different colors. <b>B.</b> Proteins were pulled down from mouse brain cytosol with the different GST fusion constructs (as indicated below <i>lanes 3–7</i>). All constructs interacted with tubulin (indicated with an <i>asterisk</i>). GST-TRESK-loop bait protein preparations contained several incompletely translated fragments in addition to the uppermost full-length product. (The molecular weight of full-length bait proteins was smaller than 47 kD in each cases.) Control assays were performed with glutathione agarose (<i>lane 1</i>) or with high amount of GST immobilized on the resin (<i>lane 2</i>). Pull-down of tubulin was much lower in these controls than in the assays containing TRESK fragments. (The lane of marker proteins was split to introduce size labels.) <b>C.</b> Proteins from the above pull-down assays (<i>even</i> lane numbers; as indicated with color coded labels below the gel) were compared to the corresponding bait protein preparations (<i>odd</i> lane numbers). Tubulin (indicated with an <i>asterisk</i>; <i>lane 2</i>, <i>4</i> and <i>6</i>) was pulled down from brain cytosol (similarly to calcineurin in the pull-down assays with fragments 174–280 and 174–247 in <i>lane 2</i> and <i>6</i>). In contrast to tubulin and calcineurin, which appeared only in the pull-down assays, some other bands were more intense in the bait protein preparations (e.g. the bacterial contaminant below 86 kD; see the <i>odd</i> lanes). Note that higher amount of bait was loaded in the control (<i>odd</i>) than in the pull-down (<i>even</i>) lanes, and the nonspecifically binding proteins of the bait preparations could also be removed by the washing steps in the pull-down assay.</p